Colorimetric Detection of Nucleic Acid Amplification

ABSTRACT

Colorimetry is used to detect amplification reaction products. A sample is contacted with a reaction mix under conditions such that an amplification reaction occurs and produces an amplification reaction product if the sample contains a target nucleic acid template molecule. The reaction mix includes an enzyme for catalyzing the amplification reaction, and at least one halochromic agent. If the target nucleic acid template molecule is present, the amplification reaction changes the starting pH of the reaction mix to cause a detectable colorimetric change of the halochromic agent, thereby indicating the presence of the target nucleic acid. If the target nucleic acid template molecule is not present, the amplification reaction does not generate an adequate number of protons to sufficiently change the starting pH of the reaction mix to cause a detectable colorimetric change of the halochromic agent, thereby indicating that the amplification reaction product has not been produced.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/359,913, filed Mar. 20, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/306,240, issued as U.S. Pat. No. 10,253,357,filed Oct. 24, 2016, which is the national stage of International (PCT)Patent Application Serial No. PCT/US2015/027556, filed Apr. 24, 2015,which claims the benefit of U.S. provisional application 61/983,687,filed Apr. 24, 2014, the contents of which are hereby incorporated byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support awarded by the SmallBusiness Innovation Research Program at the National Institutes ofHealth (Grant No. 1R430D016718-01A1). The government has certain rightsin the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Jun. 5, 2020, is namedDSS-001C2_CRF_sequencelisting.txt and is 5,646 bytes in size.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to methods and compositions for colorimetricdetection of nucleic acid amplification reaction products. Inparticular, the invention relates to accelerated colorimetric detectionof nucleic acid amplification reaction products, using a reaction mixincluding one or more halochromic agents.

Description of the Related Art

Some current methods for the detection of specific nucleic acidsequences and nucleic acid biomarkers involve fluorescence methods. DNAprimers are designed to amplify nucleic acid sequences from a sampleusing nucleic acid amplification schemes such as PCR (polymerase chainreaction) and LAMP (loop-mediated amplification). Typically, theresulting amplicons are detected and quantified through fluorescencetechniques using an intercalating fluorophore or molecular probe.However, these techniques require sophisticated instrumentation,including optical components, an excitation source, and one or moresensors for detection of the fluorescent emission. These instruments arepotentially large, cumbersome, and expensive. Alternatively, theamplicons can be colorimetrically visualized using agarose gels orlateral flow assays. However, these techniques require additional steps,which increase the time to result, and in some cases needinstrumentation such as a gel box.

SUMMARY OF THE INVENTION

Disclosed herein are methods and kits for colorimetric detection of anamplification reaction product. The methods include contacting thesample with a reaction mix under conditions such that an amplificationreaction occurs and produces an amplification reaction product if thesample contains a target nucleic acid template molecule. The reactionmix includes an enzyme for catalyzing the amplification reaction, and ahalochromic agent. In some embodiments, the reaction mix includes morethan one halochromic agent. In some embodiments, the reaction mix alsoincludes a buffer having a buffering capacity equivalent to Tris bufferat a concentration between 1 mM-19 mM in a solution having a starting pHof 8.0. If the target nucleic acid template molecule is present, theamplification reaction changes the starting pH of the reaction mix tocause a detectable colorimetric change of the halochromic agent, therebyindicating the presence of the target nucleic acid. In some embodiments,the detectable colorimetric change is quantified at a cell path lengthof 50 μm. If the target nucleic acid template molecule is not present,the amplification reaction does not generate an adequate number ofprotons to sufficiently change the starting pH of the reaction mix tocause a detectable colorimetric change of the halochromic agent, therebyindicating that the amplification reaction product has not beenproduced.

The kit includes an enzyme for catalyzing an amplification reaction, ahalochromic agent, and optionally a buffer having a buffering capacityequivalent to Tris buffer at a concentration between 1 mM-19 mM in asolution having a starting pH of 8.0. The kit further includesinstructions for use comprising instructions for contacting a samplewith a reaction mix including the buffer and the enzyme and thehalochromic agent under conditions that an amplification reaction occursand produces an amplification reaction product if the sample contains atarget nucleic acid template molecule, the reaction mix having astarting pH. If the target nucleic acid template molecule is present,the amplification reaction changes the starting pH of the reaction mixto cause a detectable colorimetric change of the halochromic agent,thereby indicating the presence of the target nucleic acid. If thetarget nucleic acid template molecule is not present, the amplificationreaction does not generate an adequate number of protons to sufficientlychange the starting pH of the reaction mix to cause a detectablecolorimetric change of the halochromic agent, thereby indicating thatthe amplification reaction product has not been produced.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 shows the DNA sequence of a template nucleic acid molecule targetregion from Schistosoma mansoni (SEQ ID NO: 23), according to anembodiment.

FIG. 2 is a graph indicating pH measurements for positive and negativeisothermal amplification reactions, according to an embodiment.

FIG. 3 is a graph showing the detection of color (hue) of positive andnegative isothermal amplification reactions at the reaction endpoints,according to an embodiment.

FIG. 4 shows the results of a gel electrophoresis assay of positive andnegative isothermal amplification reaction products, according to anembodiment.

FIG. 5 shows the normalized hue values for amplification reactions usingvarious Tris buffer concentrations, according to an embodiment.

FIG. 6 shows the normalized hue values for amplification reactions usingvarying amounts of additional hydronium ion equivalents, according to anembodiment.

FIGS. 7A, 7B, 7C, and 7D show the normalized hue values foramplification reactions using various halochromic agent concentrations,according to an embodiment.

FIG. 8 shows the compatibility of different polymerases with visualdetection of LAMP amplification, according to an embodiment.

FIGS. 9A and 9B show the normalized hue values for amplificationreactions using varying channel depths, according to an embodiment.

FIG. 10 shows the normalized hue values over time for SDA, according toan embodiment.

FIG. 11 shows the normalized hue values over time for PCR, according toan embodiment.

FIGS. 12A and 12B show the normalized contrast changes for amplificationreactions using combinations of halochromic agents, according to anembodiment.

FIG. 13 shows the normalized contrast changes over time for differentDNA template concentrations, according to an embodiment.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are compositions and methods for colorimetric detectionof nucleic acid amplification reaction products. In some embodiments,amplified reaction products are detected by a visual color changeobservation or by measuring absorbance or fluorescence of the colorchange of a halochromic agent in the amplification reaction mix.

Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The term “colorimetry” or “colorimetric” refers to techniques ofquantifying or otherwise observing colored compound concentrations insolution. “Colorimetric detection” refers to any method of detectingsuch colored compounds and/or the change in color of the compounds insolution. Methods may include visual observation, absorbancemeasurements, or fluorescence measurements, among others.

The term “halochromic agent” refers to a composition that changes colorupon some chemical reaction. In particular, a halochromic agent canrefer to a composition that changes color with a pH change. Differenthalochromic agents may change colors over different pH transitionranges.

The term “transition pH range” or “pH transition range” refers to a pHrange over which the color of a particular sample or compound changes. Aspecific transition pH range for a sample may depend on a halochromicagent in the sample (see above).

The term “nucleic acid amplification” or “amplification reaction” refersto methods of amplifying DNA, RNA, or modified versions thereof. Nucleicacid amplification includes several techniques, such as an isothermalreaction or a thermocycled reaction. More specifically, nucleic acidamplification includes methods such as polymerase chain reaction (PCR),loop-mediated isothermal amplification (LAMP), strand displacementamplification (SDA), recombinase polymerase amplification (RPA),helicase dependent amplification (HDA), multiple displacementamplification (MDA), rolling circle amplification (RCA), and nucleicacid sequence-based amplification (NASBA). The term “isothermalamplification” refers to an amplification method that is performedwithout changing the temperature of the amplification reaction. Protonsare released during an amplification reaction: for every deoxynucleotidetriphosphate (dNTP) that is added to a single-stranded DNA templateduring an amplification reaction, one proton (H⁺) is released.

The term “sufficient amount” means an amount sufficient to produce adesired effect, e.g., an amount sufficient to modulate proteinaggregation in a cell.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, thepercent “identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Compositions of the Invention

Disclosed herein are compositions and methods for accelerated andefficient colorimetric detection of nucleic acid amplification reactionproducts. In an embodiment, a colorimetric assay is used to visuallydetect the presence of an amplified nucleic acid product, whicheliminates the need for expensive and sophisticated instrumentation.

In some embodiments, the colorimetric detection of amplificationproducts is achieved by amplifying a target nucleic acid templatemolecule to obtain the amplification reaction product. The amplificationreaction includes a reaction mix. In an embodiment, the reaction mixincludes a nucleic acid template molecule, one or more enzymes forcatalyzing the amplification reaction, and one or more halochromicagents for colorimetric detection. In a further embodiment, the reactionmix also includes a buffer having a buffering capacity equivalent toTris buffer at a concentration between 1 mM-19 mM in a solution having astarting pH of 8.0. In further embodiments, the reaction mix alsoincludes a plurality of nucleic acid primers, deoxynucleotidetriphosphates (dNTPs), suitable salts for the enzyme, and othernon-buffered chemicals that enable nucleic acid amplification.

During the amplification reaction, one proton is released for each dNTPthat is incorporated into a nucleic acid template molecule. Thus, the pHof the reaction mix decreases throughout the amplification reaction. Inan embodiment, if the target nucleic acid is present, the amplificationreaction changes the starting pH of the reaction mix to cause adetectable colorimetric change of the halochromic agent, therebyindicating the presence of the target nucleic acid, and if the targetnucleic acid is not present, the amplification reaction does notgenerate a sufficient number of protons to change the starting pH of thereaction mix sufficient to cause a detectable colorimetric change of thehalochromic agent, thereby indicating that the amplification reactionproduct has not been produced. In an embodiment, the halochromic agent(or pH indicator) in the reaction mix has a transition pH range for acolorimetric change of the halochromic agent that is narrower than anexpected pH change between (1) a starting pH of the reaction mix beforethe amplification reaction is performed, and (2) an ending pH of thereaction mix after the amplification reaction has been performed.

In an embodiment, the halochromic agent is a colorimetric agent or afluorescent agent. Suitable halochromic agents include phenol red,bromocresol purple, bromothymol blue, neutral red, naphtholphthalein,cresol red, cresolphthalein, phenolphthalein, methyl red, andthymolphthalein, among others. A wide range of concentrations of thesehalochromic agents can be used in the reaction mix. Differenthalochromic agents have different transition pH ranges. In someembodiments, the halochromic agent has a transition pH range between pH5-10, between pH 6-9, or between pH 6.5-8.8. In another embodiment, thehalochromic agent is at a concentration between 25-100 μM in thereaction mix. In another embodiment, the halochromic agent is at aconcentration between 50-260 μM. In some embodiments, a combination oftwo or more halochromic agents is used in the reaction mix, whichincreases the normalized color contrast change of the reaction mix bybeing of complementary colors at the beginning and similar colors at theend of the amplification reaction. In a further embodiment, thecombination of halochromic agents comprises phenol red and bromothymolblue. In a further embodiment, the combination of halochromic agentscomprises cresol red and bromothymol blue.

In one example, Phenol red is a halochromic agent that has a transitionpH range from around 6.4-8.0. At the upper limit of the transition pHrange, phenol red is red, and at the lower limit of the transition pHrange, phenol red is yellow. A reaction mix containing phenol red willchange color from red to yellow throughout the amplification reaction,as long as the starting pH of the reaction mix is around or above 8.0,and the ending pH of the reaction mix is within the transition pH rangeor around or below 6.4.

In some embodiments, the starting pH of the reaction mix is set byadding an acid or a base to the reaction mix until the desired startingpH is reached. The ending pH of the reaction mix is determined byperforming a sample amplification reaction and measuring the ending pH(for example, with a micro-pH electrode). In an embodiment, thehalochromic agent for an amplification reaction is selected so that thetransition pH range lies in between the starting pH and ending pH. In afurther embodiment, the halochromic agent is selected so that thetransition pH range is nearer to the starting pH than the ending pH. Thehalochromic agent can also be selected based on the particular enzymeused for catalyzing the amplification reaction. Near the ending pH, theenzyme in the reaction mix terminates polymerization of theamplification reaction as the pH decreases to unfavorable H⁺concentrations. In an embodiment, additional hydronium ions or hydroniumion equivalents are added to the reaction mix via the sample. Forexample, between 4.8×10⁻⁹ and 4.8×10⁻¹⁸ additional hydronium ionequivalents per 10 μl reaction mix can be tolerated for theamplification reaction to proceed. In a further embodiment, between4.8×10⁻¹⁰ and 4.8×10⁻¹⁸, 4.8×10⁻¹² and 4.8×10⁻¹⁸, or 4.8×10⁻¹⁵ and4.8×10⁻¹⁸ can be tolerated.

Generally, the enzyme will catalyze amplification reactions within a pHrange that encompasses or is close to the transition pH range of theselected halochromic agent. Various enzymes can be used for thereaction, and different enzymes catalyze amplification reactions atdifferent pH ranges. For example, Bst polymerase is believed to catalyzeamplification reactions within the pH range of 6.6-9.0. The preferredstarting pH for Bst polymerase is greater than 7, more preferablygreater than 8.2, and more preferably at 8.8. Other examples of apreferred starting pH for Bst polymerase are found in U.S. Pat. No.5,830,714, filed Apr. 17, 1996, hereby incorporated by reference in itsentirety. In an embodiment, phenol red is coupled with Bst polymerase ina reaction mix, since the pH range at which Bst polymerase is active(6.6-9.0) encompasses the transition pH range of phenol red (6.4-8.0).In another embodiment, methyl red is coupled with U exo-Klenow fragment(polymerase for Helicase Dependent Amplification, HDA) in a reactionmix, since a starting pH at which U exo-Klenow fragment is active(around 7.5) is higher than the transition pH range of methyl red(4.8-6.2).

Other than Bst or Bst 2.0 polymerase, other enzymes capable of beingused for catalyzing the amplification reaction include the polymerasefrom Thermus aquaticus (TAQ), DNA polymerases I-IV, Kapa Polymerase, RNApolymerases I-V, T7 RNA Polymerase, a reverse transcriptase, any DNApolymerase or RNA polymerase, a helicase, a recombinase, a ligase, arestriction endonuclease, and a single-strand binding protein. In someembodiments, an isothermal amplification reaction uses an enzyme that isa strand displacement polymerase, such as phi29-DNA-Polymerase, KlenowDNA-Polymerase, Vent DNA Polymerase, Deep Vent DNA Polymerase, Bst DNAPolymerase, 9oNm™ DNA Polymerase, U exo-Klenow fragment, or mutants andvariants thereof. In some embodiments, suitable salts for the enzyme arealso added to the reaction mix. In certain embodiments, the starting pHof the reaction mix is set based on an optimal pH for the specificenzyme used for catalyzing the amplification reaction. In an embodiment,the pH of the entire DNA sample is between pH 3 and pH 11.

In other embodiments, a fluorescent halochromic agent is used to detectprotons released during amplification. The halochromic agent may changeoptical properties (such as amplitude and emitted wavelength) as the pHof the reaction mix changes during the amplification reaction.Fluorescent halochromic agents include fluorescein, pyranine, and pHrododye (Life Technologies, Carlsbad Calif.).

The base and/or acid added to the reaction mix maintains the starting pHof the reaction mix around or above an upper limit of the transition pHrange of the halochromic agent. For example, an acid such ashydrochloric acid (HCl) or sulfuric acid (H₂SO₄), or a base such assodium hydroxide (NaOH) or potassium hydroxide (KOH), can be added tothe reaction mix. In some embodiments, the acid or base sets thestarting pH of the reaction mix between pH 6-10, between pH 7-8, orbetween pH 8-8.6. In an embodiment, the reaction mix is capable ofoffsetting the starting pH of the reaction mix by less than 0.1 pHunits. In another embodiment, the reaction mix has a starting pH lowerthan 2 pH units above the upper limit of the transition pH range of thehalochromic agent. In further embodiments, the reaction mix has astarting pH lower than 1 pH unit, 0.5 pH units, or 0.1 pH units abovethe upper limit of the transition pH range of the halochromic agent. Ina further embodiment, noise from non-specific amplification is minimizedby setting the pH transition range sufficiently separated from thestarting pH of the reaction mix, so that any color change is onlyachieved by a specific and sustained amplification.

In an embodiment, the reaction mix does not require any additionalbuffering agent for the amplification reaction, since a buffering agentcould prevent large changes in pH from occurring during theamplification reaction. In another embodiment, the reaction mix containsa minimal amount of buffering agent, such that the buffering capacity ofthe reaction mixture is less than the expected change in pH duringamplification. In some embodiments, the buffer is at a concentrationbetween 1 mM and 3 mM. In a further embodiment, the buffer is at aconcentration of 1 mM. In certain embodiments, the buffer used is Trisbuffer (formulated to pH 8.8), HEPES (pH 7-9), or TAPS (pH 7-9). Inanother embodiment, the buffer used is a buffer having a bufferingcapacity equivalent to a Tris buffer at a concentration between 1 mM-19mM in a solution having a starting pH of 8.0. This broad range ofsuitable buffer concentrations allows the reaction mix to resistunwanted starting pH changes during reaction setup, unlike reactionsetups with minimal (<1 mM) Tris buffer equivalents (see U.S. Ser. No.13/799,995, filed Mar. 13, 2013). These unwanted changes in pH comeabout due to hydronium or hydroxide ion equivalents added to thereaction via the sample reagents. As colorimetric detection and enzymekinetics depend on the starting pH, the presence of buffer capacity inthe reaction mix high enough to avoid starting pH change, but low enoughto allow color change upon amplification, become important. In a furtherembodiment, the pH of the reaction mix is between pH 7.5-8.8. Table 1shows various buffers having buffering capacities equivalent to a Trisbuffer at a concentration between 1 mM-19 mM in a solution having astarting pH of 8.0. The buffer capacity (β) is defined as theequivalents of acid or base needed to change the pH of 1 Liter of bufferby 1 pH unit. This can be calculated as:β=2.3*C*(K_(a)*[H₃O⁺]/(K_(a)+[H₃O⁺])²); where C is the bufferconcentration, K_(a) is the dissociation constant for the buffer and[H₃O⁺] is the hydronium ion concentration of the buffer (which iscalculated from the reaction starting pH). The buffer capacity of 1mM-19 mM Tris (in a solution having a starting pH of 8.0) was found torange from 0.000575 to 0.010873. The starting pH of the buffer wasconsidered to be in the range of 7.5-8.8 to be compatible with thereaction biochemistry (polymerase function, nucleic acid melting, etc.).In other embodiments, the buffer has a buffering capacity equivalent toa Tris buffer at a concentration between 1.5 mM-19 mM, 2 mM-19 mM, 3mM-19 mM, 4 mM-19 mM, 5 mM-19 mM, 6 mM-19 mM, 7 mM-19 mM, or otherwise,in a solution having a starting pH of 8.0. In other embodiments, thebuffer has a buffering capacity equivalent to a Tris buffer at aconcentration between 1.92 mM-36.29 mM, 3 mM-36.29 mM, 4 mM-36.29 mM, 5mM-36.29 mM, or otherwise, in a solution having a starting pH of 8.8. Inother embodiments, the buffer has a buffering capacity equivalent to aTris buffer at a concentration between 1.48 mM-27.92 mM, 2 mM-27.92 mM,3 mM-27.92 mM, 4 mM-27.92 mM, 5 mM-27.92 mM, or otherwise, in a solutionhaving a starting pH of 7.5.

TABLE 1 Buffer Capacity Table Starting Min Conc Max Conc Buffer FullChemical Name pKa at 25° C. Reaction pH (mM) (mM) Tristris(hydroxymethyl)methy- 8.06 8.8 1.92 36.29 lamine 8.0 1.00 19.00 7.51.48 27.92 TAPS N- 8.43 8.8 1.19 22.55 Tris(hydroxymethyl)methyl- 8.01.27 23.94 3-aminopropanesulfonic 7.5 2.66 50.25 acid Bicine N,N-bis(2-8.35 8.8 1.29 24.46 hydroxyethyl)glycine 8.0 1.17 22.15 7.5 2.31 43.59Tricine N-tris(hydroxymethyl) 8.15 8.8 1.67 31.63 methylglycine 8.0 1.0319.48 7.5 1.67 31.63 TAPSO 3-[N- 7.635 8.8 4.17 78.90Tris(hydroxymethyl)methyl- 8.0 1.19 22.45 amino]-2- 7.5 1.02 19.37hydroxypropanesulfonic acid HEPES 4-(2-hydroxyethyl)-1-piperazine- 7.488.8 5.74 108.45 ethanesulfonic 8.0 1.40 26.54 acid 7.5 1.00 18.92 TESN-tris(hydroxymethyl)methyl- 7.4 8.8 6.79 128.39 2-aminoethanesulfonic8.0 1.56 29.46 acid 7.5 1.01 19.16 MOPS 3-(N- 7.2 8.8 10.46 197.77morpholino)propanesulfonic 8.0 2.12 40.03 acid 7.5 1.12 21.26 PIPES 1,4-6.76 8.8 27.91 500.00 piperazinediethaiiesulfonic 8.0 4.86 91.88 acidacid 7.5 1.92 36.29 SSC Saline Sodium Citrate 7.0 8.8 16.28 300.00 8.03.03 57.20 7.5 1.37 25.90

In an embodiment, a magnesium compound is added to the reaction mix,because magnesium promotes nucleotide incorporation into the templateand influences the activity of the polymerase. In a further embodiment,the concentration of a magnesium compound (such as magnesium sulfate) inthe reaction mix is at least 0.5 mM, at least 1 mM, at least 2 mM, or atleast 4 mM. In an embodiment, the concentration of added magnesium ionis dependent on the concentration of dNTPs, nucleic acid template, andprimers. In an embodiment, the ratio of dNTPs to magnesium sulphate inthe reaction mix is less than 1:2, less than 1:3, less than 1:4 or lessthan 1:5.

In some embodiments, monovalent cations are added to the reaction mix.Monovalent cations include potassium, ammonium, and quaternary ammonium,among others.

Monovalent cations can affect the melting characteristics of the nucleicacid template and improve the efficiency of the enzyme. In anembodiment, potassium is in the reaction mix at a concentration of lessthan 50 mM, or less than 15 mM. In another embodiment, quaternaryammonium salts are in the reaction mix at a concentration of greaterthan 2 mM, greater than 5 mM, or greater than 8 mM. In anotherembodiment, an ammonium compound (such as ammonium chloride) is in thereaction mix at a concentration of less than 15 mM, or less than 10 mM.Ammonium (NH₄ ⁺) has some buffering capability, thus the finalconcentration of ammonium compounds in the reaction mix should beminimized while maintaining optimal amplification yield.

In an embodiment, the concentrations of other reagents of the reactionmix are kept at amounts as generally used in amplification reactions.See Notomi T et. al. Nucleic Acids Res. 2000 Jun. 15; 28(12): E63;Nature Protocols 2008, Loop-mediated isothermal amplification (LAMP) ofgene sequences and simple visual detection of products, 2008 3(5): pg880, hereby incorporated by reference in its entirety. In an embodiment,the Bst or Bst 2.0 enzyme is used, and the amount of enzyme is at least0.8 Unit per microliter of combined fluid. In this embodiment, Betaineis also present in the reaction mix at a concentration between 0-1.5 Mor 0.8M-1 M, and the total concentration of primers is between 3.6 μMand 6.2 μM. In some embodiments, any of the following reagents ispresent in the reaction mix: Tris buffer (pH 8.8) at 20 mM, KCl at 10mM, MgSO₄ at 8 mM, (NH₄)₂SO₄ at 10 mM, Tween 20 at 0.1%, Betaine at 0.8M, dNTPs at 1.4 mM each, MnCl₂ at 0.5 mM, FIP at 1.6 μM, F3 at 0.2 μM,B3 at 0.2 μM, primers at a total concentration of 5.2 μM(2*(1.6+0.8+0.2), and Bst/Bst 2.0 at 8 U per 10 μL.

The above reagent concentrations have been found to provide goodamplification yield and low buffering capacity so that a halochromic pHsensor can be used to detect protons released during the amplificationreaction. In some embodiments, the concentrations of reaction mixreagents depend on the enzyme selection. In further embodiments,guidance regarding appropriate reagent concentrations is available fromthe enzyme manufacturers. In an embodiment, the ratio of the samplevolume to the reaction mix volume is such that the sample is dilutedbetween 5% and 40% when the reaction mix is added.

In some embodiments, amplification reaction reagents are storedseparately before being added to a reaction mix, since some reagentshave specific required conditions for stability. For example, the enzymemay be stored long term in a moderately buffered solution separate fromthe other reagents to ensure stability of the enzyme. Upon mixing withthe remaining reagents in the reaction mix, the buffering agent becomessufficiently diluted so as not to significantly mask a pH change. Inaddition, primers for specific genes of interest may be provided in aseparate solution or in a lyophilized form.

In some embodiments, the amplification reaction is performed within amicrotube. In other embodiments, the amplification reaction is performedwithin a fluidic or microfluidic structure. In some embodiments, thefluidic or microfluidic structure is a well, chamber, or channel thatreceives the reagents and the nucleic acid sample separately, and thenmixes the components together. In another embodiment, the fluidic ormicrofluidic structure is a well, chamber, or channel that receives thepre-mixed reaction mix. In a further embodiment, the fluidic ormicrofluidic structure possesses a long optical path for colorimetricobservation, or a fluorescent/absorbance excitation source and detector.In another embodiment, the fluidic or microfluidic structure receivesthe reagents in a lyophilized form, and subsequently receives thenucleic acid sample and hydration solution. In an embodiment, a chamberfluidic or microfluidic structure has a channel depth ranging between 50μm-400 μm or greater. In a further embodiment, colorimetric observationis accomplished for channel depths (path length) of 50 μm, 50 μm-400 μm,or 50 μm or greater.

Some embodiments include a kit for colorimetric detection of anamplification product. The kit may include one or more halochromicagents, one or more enzymes for catalyzing an amplification reaction,and instructions for contacting a sample with a reaction mix includingthe buffer and the enzyme and the halochromic agent under conditionsthat an amplification reaction occurs and produces an amplificationreaction product if the sample contains a target nucleic acid templatemolecule, the reaction mix having a starting pH, and if the targetnucleic acid template molecule is present, the amplification reactionchanges the starting pH of the reaction mix to cause a detectablecolorimetric change of the halochromic agent, thereby indicating thepresence of the target nucleic acid, and if the target nucleic acidtemplate molecule is not present, the amplification reaction does notgenerate a sufficient number of protons to change the starting pH of thereaction mix sufficient to cause a detectable colorimetric change of thehalochromic agent, thereby indicating that the amplification reactionproduct has not been produced. In another embodiment, the instructionsare for contacting a nucleic acid template molecule with the halochromicagent and enzyme in a reaction mix, under conditions that result in (1)an amplification reaction that amplifies the nucleic acid templatemolecule to produce an amplification reaction product, and (2)generation of a sufficient number of protons so that an ending pH of thereaction mix is sufficiently low to produce a detectable colorimetricchange of the halochromic agent, thereby indicating that theamplification reaction product has been produced. In furtherembodiments, the kit also includes an acid or base, dNTPs, primers, andmonovalent cations. In a further embodiment, the kit includes thefollowing reagents at the following concentrations:

-   -   Bst or Bst 2.0 polymerase, at least 0.8 Unit per microliter;    -   Betaine at 0.8 M;    -   Primers at 3.6 μM total;        -   FIP and BIP primers at 1.6 μM        -   F3 and B3 at 0.2 μM    -   Magnesium sulfate at 8 mM;    -   Ammonium sulfate at 10 mM;    -   Potassium chloride at 10 mM;    -   Sodium hydroxide to set the starting pH of the reaction mix;    -   Tween20 at 0.1%;    -   dNTP's at 1.4 mM each;    -   Phenol red at 50 μM.        In a further embodiment, the kit includes LoopF and LoopB        primers at 0.8 μM each.

Methods of the Invention

The amplification reaction amplifies nucleotides from a nucleic acidtemplate. In some embodiments, the amplification reaction is anisothermal amplification reaction, such as a strand displacementreaction. In a further embodiment, a strand displacement reaction isprovided by a polymerase with strand displacement activity underreaction conditions such that strand displacement is possible. Examplesof strand displacement reactions include strand displacementamplification (SDA), multiple displacement amplification (MDA), rollingcircle amplification (RCA) or loop mediated isothermal amplification(LAMP). In other embodiments, the amplification reaction includes othernon-isothermal amplification reactions such as polymerase chain reaction(PCR).

In certain embodiments, the amplification reaction performed is LAMP. Ina LAMP reaction, a double- or single-stranded DNA template in dynamicequilibrium at an elevated temperature is amplified using two or threepairs of primers. The primers are designed based on the DNA template,using primer design software such as LAMP Designer (Premier Biosoft,Palo Alto, Calif.). In the first step of the LAMP reaction, the F2region of the FIP (Forward Inner Primer) anneals to the single strandedDNA at the respective complementary (F2c) position. Next, a polymerasewith strand displacement activity incorporates dNTPs along the templatefrom the 3′ end of F2. The incorporation of nucleotides releasesprotons, reducing the pH of the reaction mix. Then, the F3 forwardprimer anneals to the F3c region upstream of the F2 region and on thetemplate. The F3 forward primer begins amplifying the template strand,which releases further protons and displaces the FIP-incorporated strandthat was synthesized previously. This single strand contains an F1sequence (within the target sequence) along with its complementary F1csequence (within the FIP). This forms a stem-loop as F1c anneals to F1at the 5′ end. At the same time, the BIP (Backward Inner Primer) annealsto the other end of the strand and nucleotides extend from B2, releasingmore protons. The backward primer B3 then binds to the B3c region,downstream of the B2 region, displaces the BIP-amplified strands andpromotes extension to create the double strand. This displaced strandnow contains a B1 sequence (within the target sequence) along with itscomplementary B1c sequence (within the BIP), forming another stem loopin the 3′ end. The structure now has two stem-loop structures at eachend from which continuous displacement and extension occur to amplifythe template. The LAMP reaction can be amplified by adding furtherForward and Backward Loop primers to produce more amplicons with stemloop structures.

The LAMP procedure can take place at a fixed temperature, minimizing theneed for any expensive thermocycling equipments. Typically, isothermalmethods require a set temperature, which is determined by the selectedreagents. For example, enzymes function best between 60-65° C. in LAMPmethods.

Colorimetric detection of the nucleic acid amplification reactionproduct can be performed in real-time throughout the amplificationreaction, or after the performance of the amplification reaction.Detection of the colorimetric change of the reaction mix can beassociated with a digital indication of a presence or absence of theamplification reaction product. In other words, a visual observation ofthe color change of the reaction mix can provide information regardingwhether the amplification reaction product is present or absent. Incertain embodiments, detection of a colorimetric change of the reactionmix indicates that the exponential or plateau phase of the amplificationreaction has been obtained.

In some embodiments, detection of the amplification reaction product isaccelerated relative to an amplification reaction that uses a reactionmix without a halochromic agent. In further embodiments, thecolorimetric change of the reaction mix is detected in less than 60minutes from a starting time of the amplification reaction. Accelerateddetection of the amplification reaction product is obtained because thehalochromic agent (a weak acid or base) in the reaction mix absorbsprotons generated during the amplification reaction, and recombinationof the free protons acts to accelerate the detection of theamplification reaction. The reaction can be designed so that minimalamplification is required to generate a pH transition sufficient for thehalochromic agent to change color. Conventional amplification techniquesthat use fluorescent intercalating dyes, molecular beacons,hybridization probes, dye-based detection, UV-Vis, or other detectionmethods require a certain threshold amount of amplification to occurbefore an amplification signal is detectable. However, the methods ofthe present invention require a relatively smaller threshold amount ofamplification before a color change of the halochromic agent isdetectable, and therefore the detection of an amplification reactionproduct is accelerated relative to conventional amplification methods.

In some embodiments, the amplification reaction product is detectedvisually by observation of a color change of the reaction mix. In afurther embodiment, the human eye is used for the visual detection. Inanother embodiment, a camera, a computer, or some other optical deviceis used for the visual detection or for imaging the reaction mix.Imaging programs include Photoshop (Adobe, San Jose Calif.), ImageJ(National Institutes of Health, Bethesda Md.), and MATLAB (MathWorks,Natick Mass.). In another embodiment, the amplification reaction productis detected by measuring fluorescence of the reaction mix, usingfluorescence spectroscopy methods. In another embodiment, theamplification reaction product is detected by measuring absorbance ofthe reaction mix, using absorption spectroscopy methods. In a furtherembodiment, the endpoint or overall change in absorbance or fluorescenceof the reaction mix is measured at a given wavelength or set ofwavelengths.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1: Colorimetric Detection of a Nucleic Acid AmplificationReaction Product

In an assay for colorimetric detection of a nucleic acid amplificationreaction product, the following reagents were mixed together to producea 2× reagent mix:

-   -   Magnesium Sulphate (Sigma Aldrich) at 16 mM    -   Ammonium Sulphate (Sigma Aldrich) at 20 mM    -   Potassium Chloride (Sigma Aldrich) at 20 mM    -   Sodium hydroxide (Sigma Aldrich) at a concentration that sets        the starting pH of the reagent mix to 8.8 pH

The reagent mix was adjusted to an initial pH of 8.8 to enable efficientinitial polymerization. The reagent mix was autoclaved for 1 hour forsterilization. The following ingredients were then added (in a sterileform) to the reagent mix to generate the reaction mix:

-   -   Tween20 (Sigma Aldrich) at 0.1% (v/v)    -   dNTPs (NEB) at 1.4 mM each    -   Phenol Red (Sigma Aldrich) at 50 μM    -   Bst polymerase (NEB) at 0.8 Unit per microliter (the enzyme        storage buffer contributing 1 mM Tris buffer, 5 mM KCl, 0.01 mM        EDTA, 0.1 mM DTT, 0.01% Triton X-100 (v/v) and 5% Glycerol        ((w/v) to the reaction mix)    -   Betaine (Sigma Aldrich) at 0.8 M

Primers and a nucleic acid template were added to the reaction mix. Theprimers were designed for LAMP and included two pairs of primers(solubilized in 1× Tris EDTA buffer) at a total concentration of 3.6 μMas described above. Primer F3 has the sequence: GATCTGAATCCGACCAACCG(SEQ ID NO: 1); primer B3 has the sequence: AACGCCCACGCTCTCGCA (SEQ IDNO: 2); the primer FIP has the sequence:AAATCCGTCCAGTGGTTTTTTTGAAAATCGTTGTATCTCCG (SEQ ID NO: 3); and the primerBIP has the sequence: CCGAAACCACTGGACGGATTTTTATTTTTAATCTAAAACAAACATC(SEQ ID NO: 4). The nucleic acid template molecule was purified fromSchistosoma mansoni. FIG. 1 shows the SM1-7 target region of the nucleicacid template molecule (see Hamburger et al, Detection of Schistosomamansoni and Schistosoma haematobium DNA by Loop-Mediated IsothermalAmplification: Identification of infected Snails from Early Prepatency,Am J Trop Med Hyg, 2010). The positive test reactions contained templateDNA, and the negative control reactions contained water. The reactionmixes had a starting pH in the range of 7.5-8.5. The reaction mixes wereheated in micro-tubes to 63° C. on a thermocycler to allow templateamplification. After a predetermined reaction period of 45 minutes,during which sufficient template amplification occurred, the resultantcolor of the reaction mix was visually observed.

During the amplification process, the pH of the reaction mix was reducedfrom 7.5-8.5 to around 6.6 in a repeatable fashion. FIG. 2 is a graphshowing the pH measurements for repeated positive (test) and negative(negative control) amplification reactions. The halochromic agent usedwas Phenol red, which has a transition pH range of 6.8-8.2. Phenol redchanges color over this transition pH range from red to yellow (when thepH is lowered from the upper pH limit to the lower pH limit). In theassay, the reaction mix changed color from red (at pH 8.0) to yellow (atpH 6.6) in response to the pH change during nucleic acid amplification.FIG. 3 is a graph showing the difference in contrast value using HSV(hue, saturation, value) of images of the reaction mixes of a positiveand negative amplification reaction at the reaction endpoints. The colorchange is quantitatively demonstrated in the hue variable. To confirmthat the color change was due to target DNA amplification, endpointreactions were analyzed using gel electrophoresis to verify the presenceof amplicons (FIG. 4).

Using this method, amplification of a DNA template can be easilyobserved, either at the reaction end-point or in real-time throughoutthe reaction, by visually observing the color change in the reactionmix, or by measuring the absorbance or fluorescence of the reaction mix.This mechanism generates much larger contrast in comparison to othercolorimetric detection techniques and can be imaged without the need ofexpensive optical instrumentation.

Example 2: Detection of LAMP Amplification Using a Visual HalochromicAgent

LAMP reactions were performed with a reaction mix comprising of: 10 mM(NH4)₂SO4, 15 mM KCl, 0.1 mM EDTA, 0.1 mM DTT, 0.01% Triton X-100 (v/v),5% Glycerol, 8 mM MgSO₄, 1.4 mM each dNTPs, 0.1% v/v Tween-20, 0.8 MBetaine. Three primer pairs, specific to different targets, were addedto a final concentration of 1.6 μM each for FIP/BIP, 0.2 μM each forF3/B3, 0.4 μM each for LoopB/F. The final reaction volume is 10 μL andwas held at 63° C. for different incubation times.

In FIG. 5, the final Tris buffer concentration of the reaction mix wasvaried from 0.34 mM to 19 mM (by varying amount of Tris bufferformulated to pH 8.8). Reactions were performed with primers for lambdaphage DNA, 5 ng of lambda DNA (New England Biolabs), 0.8 U/μl Bst 2.0DNA polymerase (New England Biolabs) and 0.2 mM Neutral Red (SigmaAldrich). The reaction tubes were then imaged and the Normalized Huevalue was calculated for the color of the reaction mix. The NormalizedHue value was defined as the difference in Hue values between a positiveand a no-template negative reaction. A color change, indicated by achange in the Normalized Hue value above the visualization threshold(dotted line), was observed for buffer concentrations as high as 19 mMTris. This indicates that reaction mix with buffer capacities equivalentto >1 mM and <19 mM Tris allow enough pH change for visual color changedetection.

In FIG. 6, the tolerance of this visual detection method to excesshydronium ions added to the reaction mix was evaluated. This toleranceis important to allow the use of a wide variety of DNA samples which canadd a range of hydronium or hydroxide ion equivalents to the reaction.Reactions were performed with 2 mM final Tris buffer concentration, 5 nglambda DNA target, 0.8 U/μL Bst DNA polymerase and 0.2 mM Neutral Redhalochromic agent. The change in Normalized Hue value indicates thatthis visual detection chemistry works with 4.8×10⁻⁹ till 4.8×10⁻¹⁸additional hydronium ion equivalent per 10 uL reaction.

In FIGS. 7A-7D, the compatibility of different pH indicators andamplification targets with visual detection of LAMP amplification wasevaluated. The reactions were performed with final Tris bufferconcentration in the range of 1.2-1.3 mM and 0.8 U/pt Bst DNApolymerase. Three different indicator were tested with 5 ng lambda DNAtarget: 50 μM Phenol Red, 260 μM Cresol Red and 160 μM Bromothymol Blue(FIG. 7A). High contrast change in the normalized hue value was observedfor all indicators tested.

Concentration sweeps were also performed for these indicatorsBromothymol Blue (FIG. 7B top left), Cresol Red (FIG. 7B top right),Neutral Red (FIG. 7B bottom left) and Phenol Red (FIG. 7B bottom right)with Lambda target, which demonstrated the wide range of concentrationsthat are compatible with the chemistry. LAMP assays using 130 ngSchistosoma mansoni gDNA with 50 μM Phenol Red (FIG. 7C) and Human GAPDHmRNA with 0.2 mM Neutral Red (FIG. 7D) were also tested visual detectionof these targets was demonstrated at end-point.

In FIG. 8, the compatibility of different polymerases with visualdetection of LAMP amplification was evaluated. The reactions wereperformed with 1.3 mM final Tris buffer concentration, 5 ng lambda DNAtarget and 0.2 mM Neutral Red. 0.8 U/μl of two different polymerases,Bst 2.0 and Gspm 2.0 (OptiGene), were used. High contrast color changewas observed for both polymerases after 60 minutes of incubation (FIG.8).

TABLE 2 Sequences Used Lambda FIP SEQ ID NO: 5 Lambda BIP SEQ ID NO: 6Lambda F3 SEQ ID NO: 7 Lambda B3 SEQ ID NO: 8 Lambda Loop F SEQ ID NO: 9Lambda Loop B SEQ ID NO: 10 Schistosoma F3 SEQ ID NO: 1 Schistosoma B3SEQ ID NO: 2 Schistosoma FIP SEQ ID NO: 3 Schistosoma BIP SEQ ID NO: 4GAPDH F3 SEQ ID NO: 11 GAPDH B3 SEQ ID NO: 12 GAPDH FIP SEQ ID NO: 13GAPDH BIP SEQ ID NO: 14 GAPDH Loop F SEQ ID NO: 15 GAPDH Loop B SEQ IDNO: 16

Example 3: Visual Detection of LAMP Amplification in Sub-Millimeter PathLengths

LAMP reactions were performed as in Example 1 with 1.3 mM final Trisbuffer concentration (buffer formulated to pH 8.8), 0.8 U/μl of Bst 2.0DNA Polymerase, 5 ng lambda DNA template and 0.2 mM Neutral Red or 160μM Bromothymol Blue. Both the positive and the no-template negativereactions were added after amplification to flow chambers with varyingchannel depths (FIG. 9A for Neutral Red and FIG. 9B for BromothymolBlue). These flow chambers were machined in acrylic with channel depthsranging from 50 μm to 400 μm. High contrast color difference (above thevisual detection threshold; dotted line) between the positive and thenegative reactions was observed for channel depths of 50 μm and above.This demonstrates that this visual detection chemistry is amenable foruse in reaction chambers with sub-millimeter path lengths (depths) andabove. Such reaction chambers can be used to reduce the amount ofreagents used and to allow multiple reactions to take place in a certainfootprint (eg. in a microfluidic cartridge).

Example 4: Detection of Strand Displacement Amplification (SDA) Using aVisual Halochromic Agent

SDA reactions were performed using a reaction mix comprising of: 1.3 mMfinal Tris buffer concentration (buffer formulated to pH 8.8), 10 mM(NH4)₂SO4, 50 mM KCl (adjusted to pH 8.5), 8 mM MgSO₄, 4.4 mM each dATP,dGTP, dTTP, 0.8 mM dCTP-aS (TriLink Biotechnologies), 0.1% v/v Tween-20,0.8 M Betaine, 0.32 U/μl Bst DNA polymerase (New England Biolabs), 0.2U/uL BSoBI (New England Biolabs) and 0.2 mM Neutral Red halochromicagent. Primers designed for human BRCA1 (SDAf: SEQ ID NO: 17; SDAr: SEQID NO: 18; BF: SEQ ID NO: 19; BR: SEQ ID NO: 20) were added to thereaction at 0.5 μM final concentration each. 5 ng of HeLa gDNA was addedto a final reaction volume of 25 μL and was held at 65° C. for differentincubation times. A change in Normalized Hue value over time (FIG. 10)indicates that this visual detection chemistry works with SDA.

Example 5: Detection of PCR Amplification Using a Visual HalochromicAgent

PCR reactions were performed using a reaction mix comprising of: 50 mMKCl and 2 mM MgCl₂ (pH adjusted 8.5), 0.5 mM each dNTP, 5 U Taq DNApolymerase (New England Biolabs) and 0.2 mM Neutral Red halochromicagent. Total carry-over Tris-HCl concentration from enzyme storagebuffer and primers (Forward: SEQ ID NO: 21; Reverse: SEQ ID NO: 22) was1.15 mM in the final reaction mix. Primers were designed for Escherichiacoli 16s rRNA gene and added to the reaction at 0.5 μM finalconcentration each. 10 ng of E. coli gDNA was added to a final reactionvolume of 25 μL and was initially held at 95° C. hold for 2 min,followed by 50 cycles of 95° C. for 10 sec, 55° C. for 30 sec, 68° C.for 30 sec. A change in Normalized Hue value over time (FIG. 11)indicates that this visual detection chemistry works with PCR.

Example 6: Increase in Visual Detection Contrast with Combination ofHalochromic Agents

LAMP reactions were performed as in Example 1 with 1.3 mM final Trisbuffer concentration (buffer formulated to pH 8.8), 0.8 U/μl of Bst 2.0DNA Polymerase and 5 ng lambda DNA template. The color change contrastwas evaluated for Phenol Red at 50 μM concentration and combination ofPhenol Red and Bromothymol Blue at 50 μM and 160 μM concentrationsrespectively (FIG. 12A). The color change contrast was also evaluatedfor Cresol Red at 260 μM concentration and combination of Cresol Red andBromothymol Blue at 260 μM and 160 μM concentrations respectively (FIG.12B). The contrast values were calculated from the RGB values of imagesof the reaction mix using the formula: 0.299R+0.587G+0.114B. Thenormalized contrast change was defined as the difference betweenpositive and negative reaction contrast values normalized to thebackground. The increase in the normalized contrast change with the useof the halochromic agent combination demonstrates the utility of suchcombinations.

Example 7: Real-time Color Monitoring of Amplification forQuantification

Using Visual Halochromic Agents

LAMP reactions were performed as in Example 1 with 1.3 mM final Trisbuffer concentration (buffer formulated to pH 8.8), 0.8 U/μl of Bst 2.0DNA Polymerase, Phenol Red and Bromothymol Blue at 50 μM and 160 μMconcentrations respectively and varying lambda DNA templateconcentrations. Color change contrast was evaluated for lambda DNAtarget at 0.5 fg/μl, 0.05 pg/μl and 0.5 pg/μl final concentrations. Thecontrast values were calculated from the RGB values of images of thereaction mix as described in Example 5. The results (FIG. 13) indicatethat the higher DNA concentrations led to a detectable change in visualcontrast earlier than the lower DNA concentrations. Hence, wedemonstrate the ability to distinguish between different targetconcentrations with the real-time color monitoring of this chemistry.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

1. A composition of matter for colorimetric detection of anamplification reaction product in a sample, the composition of mattercomprising a reaction mix comprising: a buffer comprising one or morebuffering agents, the buffer having a net buffering capacity equivalentto Tris buffer at a concentration between 1.5 mM-19 mM in a solutionhaving a pH of 8.0; an enzyme for catalyzing an amplification reaction;and a colorimetric agent having a transition pH range between a startingpH of the reaction mix and an expected ending pH of the reaction mix,the expected ending pH of the reaction mix affected by the amplificationreaction.
 2. The composition of matter of claim 1, wherein acolorimetric change of the colorimetric agent caused by theamplification reaction is capable of being quantified at a cell pathlength of 50 μm.
 3. The composition of matter of claim 1, wherein thereaction mix comprises at least two colorimetric agents.
 4. Thecomposition of matter of claim 1, wherein detection of the amplificationreaction product is accelerated relative to an amplification reactionusing a reaction mix without a colorimetric agent, and wherein theamplification reaction using a reaction mix without a colorimetric agentcomprises detection of amplification reaction product by fluorescentintercalating dyes, molecular beacons, hybridization probes, UV-Vis,Agarose Gels or Lateral Flow Assay.
 5. The composition of matter ofclaim 1, wherein the amplification reaction is an isothermal reaction,such as a strand displacement amplification, a multiple displacementamplification, a recombinase polymerase amplification, a helicasedependent amplification, a rolling circle amplification, or a loopmediated isothermal amplification.
 6. The composition of matter of claim1, wherein the colorimetric agent is at a concentration between 50μM-260 μM.
 7. The composition of matter of claim 1, wherein a solutioncontaining a nucleic acid template molecule of the sample is capable ofoffsetting the starting pH of the reaction mix by less than 0.1 pH unitsand wherein the solution contributes between 4.8×10⁻⁹ to 4.8×10⁻¹⁸hydronium ion equivalents to the reaction mix, per 10 uL reaction mix.8. The composition of matter of claim 1, further comprising an acid orbase.
 9. The composition of matter of claim 1, further comprising atleast one of: dNTPs, primers, and a monovalent cation.
 10. Thecomposition of matter of claim 1, wherein the enzyme is a reversetranscriptase, a DNA polymerase, a RNA polymerase, an RNase, a helicase,a recombinase, a ligase, a restriction endonuclease, a TAQ polymerase,or a single-strand binding protein.
 11. The composition of matter ofclaim 1, wherein the colorimetric agent comprises one or more of phenolred, bromocresol purple, bromothymol blue, neutral red,naphtholphthalein, cresol red, cresolphthalein, and phenolphthalein. 12.The composition of matter of claim 1, wherein the transition pH of thecolorimetric agent is between pH 5-10.
 13. A composition of matter forcolorimetric detection of an amplification reaction product in a sample,the composition of matter comprising a reaction mix comprising: a buffercomprising one or more buffering agents, the buffer having a netbuffering capacity equivalent to Tris buffer at a concentration between1.5 mM-19 mM in a solution having a pH of 8.0; an enzyme for catalyzingan amplification reaction; and a halochromic agent.
 14. The compositionof matter of claim 13, wherein detection of the amplification reactionproduct is accelerated relative to an amplification reaction using areaction mix without a halochromic agent, and wherein the amplificationreaction using a reaction mix without a halochromic agent comprisesdetection of amplification reaction product by fluorescent intercalatingdyes, molecular beacons, hybridization probes, dye-based detection,UV-Vis, Agarose Gels or Lateral Flow Assay.
 15. The composition ofmatter of claim 13, wherein the halochromic agent is at a concentrationbetween 50 μM-260 μM.
 16. The composition of matter of claim 13, whereina solution containing a nucleic acid template molecule of the sample iscapable of offsetting a starting pH of the reaction mix by less than 0.1pH units and wherein the solution contributes between 4.8×10⁻⁹ to4.8×10⁻¹⁸ hydronium ion equivalents to the reaction mix, per 10 uLreaction mix.
 17. The composition of matter of claim 13, furthercomprising at least one of: dNTPs, primers, and a monovalent cation. 18.The composition of matter of claim 13, wherein the enzyme is a reversetranscriptase, a DNA polymerase, a RNA polymerase, an RNase, a helicase,a recombinase, a ligase, a restriction endonuclease, a TAQ polymerase,or a single-strand binding protein.
 19. The composition of matter ofclaim 13, wherein the halochromic agent has a transition pH rangebetween a starting pH of the reaction mix and an expected ending pH ofthe reaction mix, the expected ending pH of the reaction mix affected bythe amplification reaction.
 20. The composition of matter of claim 19,wherein the transition pH of the halochromic agent is between pH 5-10.